Our laboratory has been, for over last 15 years, engaged in molecular studies related to the host-pathogen interaction during M. tuberculosis infection. More pointedly, the identification of biomarkers of mycobacterial infection, consequent to mycobacteria and MΦ interaction, in vitro, has been our major focus. In the present studies, our most important and striking observation has been that mycobacteria, irrespective of their viability, virulence and pathogenicity, following their interaction with the PMs, in vitro, induced them to elaborate significantly (p < 0.05) enhanced levels of IL-6 only; the enhancement of the elaboration of other studied cytokines (IL-1β, IL-10, IL-12 p40, and IL-12 p70) was not significant. Because a 5-plex cytokine array (IL-1β, IL-6, IL-10, IL-12p40 and IL-12p70) was used, it is possible that other cytokines, if up-regulated, would have missed quantification. However, literature survey has shown that in most of such studies including those using human MΦs, mainly TNF, IFN-γ and GM-CSF are up-regulated, albeit not the extent we have observed enhanced IL-6 elaboration. Nevertheless, future studies using larger plex cytokine arrays may shed more light on this point. Therefore, our results, for the first time, report that IL-6 appears to be the only cytokine whose elaboration is enhanced significantly (p < 0.05) following the interaction of PMs with mycobacteria, in vitro.
In the recent past, a great significance has been attached to the identification of biomarkers, also known as biological indicators, of TB. According to Parida and Kaufmann (2010) a biomarker can be defined as a “characteristic that is objectively measured and evaluated as an indicator of a physiological or pathological process or pharmacological response(s) to a therapeutic intervention” (Parida and Kaufmann, 2010). However, so far, no biomarker(s) has been identified that can be used effectively for TB. This probably is one good reason that we still do not have suitable drugs, vaccines and diagnostic tests for TB. Therefore, there is an urgent need to have a suitable biomarker or a group of markers (also called as a biosignature) that is a true statistical reflection and is pathophysiologically related to the clinical outcome of TB, with or without therapeutic intervention. In the present study, apparently for the first time, an effort has been made to project the elaboration of IL-6, as a sequel to the interaction of PMs with mycobacteria, in vitro, as a potential biomarker of mycobacterial infection, either stand-alone or along with other cytokines, which may be developed further as a biomarker/biosignature of TB.
The concept of cytokines to function as biomarkers is well established and has been reviewed recently (Doherty et al., 2009), and it has been suggested that both IFN-γ and TNF-α can function as excellent biomarkers for the clinical assessment of TB, as their expression and elaboration has been thought to have important bearing on the immunopathogenesis of TB. Additionally, as both of these cytokines are induced and expressed rapidly early in the infection, they can be detected and quantified much before the onset of the symptoms of TB. Because IFN-γ is induced in a pathogen-specific manner, it thus constitutes a preferred biomarker. IL-4 mRNA expression has been reported to increase in healthy individuals who later develop active TB, and decrease soon after the treatment. Wassie et al. (2008) have reported that in TB patients mRNA expression for IL-4δ2, an IL-4 antagonistic splice variant, appeared to be similar to that of IFN-γ. Rhodes et al. (2007) have reported that there is a strong possibility that the ratio of the levels of IL-4 and IL-4 δ2 may correlate with the severity of TB, and thus may serve as a true reflection of the disease. This contention is further supported by a recent report (Siawaya et al., 2008), which indicates that quick changes in the ratio of IL-4 and IL-4δ2 levels during TB treatment may serve as good indicators of further treatment outcome. Augmented IL-6 elaboration can occur during various infections or inflammation. C-reactive protein, an APP, and other markers of inflammation have also been suggested to function as biomarkers of TB (Doherty et al., 2009). IL-6 is a known inducer of APPs, and Singh and Kaur (2006) have reported a near parallel increase in both serum amyloid P-component (SAP), an APP in mice, and IL-6, in M. tuberculosis-infected mice. Our results which demonstrate the elaboration of only IL-6 as the major cytokine following the interaction of PMs with mycobacteria, in vitro, also seem to be in consonance with earlier reports (Mattos et al., 2010; El-Ahmady et al., 1997). Therefore, IL-6 can be expected to be developed as a potential biomarker of TB, either stand-alone or along with other cytokines.
The mechanisms by which M. tuberculosis and M. smegmatis induce MΦs to elaborate of IL-6 are not yet properly understood. However, the lipoarabinomannan (LAM) present in the M. tuberculosis cell wall, independent of lipopolysaccharide, in a dose-dependent manner, has been reported to induce MΦs to elaborate IL-6, in vitro (Zhang et al., 1994). The LAM-induced mycobacterial response elements (transcription factors) NF-IL6 and NF-kB on the IL-6 gene, following their binding with the IL-6 regulatory region, regulate the expression of IL-6 gene (Zhang et al., 1994). A similar molecular mechanism(s) of IL-6 induction may also be operational in our studies reported herein.
We have observed that of all the cytokines studied, invariably, only IL-6 elaboration was enhanced significantly (p < 0.05). Though, apparently, there is no explanation for these observations, the following reports appear supportive. Denis (1992) has demonstrated that in tissue culture medium, which does not support full growth, both human and mouse rIL-6 function as potent growth factors for four virulent strains of M. avium; IL-6 was rapidly taken-up by the mycobacteria through a single receptor species of 50 nM Kd with approximately 15, 000 receptors/mycobacterium. It may just be possible that mycobacteria, including M. tuberculosis, may induce the augmented elaboration of IL-6 to support their own growth. Presently, our lab is engaged in the generation of more specific evidences to this effect.
The PMs infected with live M. tuberculosis H37Ra, as compared to those infected with M. tuberculosis H37Rv and M. smegmatis, invariably, elaborated significantly (p < 0.001) enhanced levels of IL-6. A major difference exists between phagosomes containing pathogenic and non-pathogenic mycobacteria. Phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid, is essential for the phagosomal acquisition of lysosomal contents (Vergne et al., 2005). M. tuberculosis H37Rv secrets a lipid phosphatase called SapM, which hydrolyzes PI3P and thus inhibits the fusion of phagosomes with late endosomes (Saleh and Belisle, 2000). Non-pathogenic mycobacteria, on the other hand, do not secrete SapM, and thus no inhibition of the fusion of phagosomes with late endosomes occurs. Because phagosome-lysosme fusion is known to result in enhanced elaboration of cytokines, it may be the only possible reason or one of the possible reasons for the augmented elaboration of cytokines by non-pathogenic mycobacteria. Another reason for the differential elaboration of cytokines by pathogenic and non-pathogenic mycobacteria may be because of the reported difference in their LAM structure. LAM from non-pathogenic mycobacteria is characterized by its extensive arabinose side-chains, and is an extremely potent inducer of cytokines; paradoxically, LAM from pathogenic mycobacterial species, in which short mannan segments extensively mask the arabinan side chain, is approximately 100-fold less potent (Chatterjee et al., 1992). The differences in the LAM structure have also been associated with the ability of mycobacteria to survive and replicate within MΦs, and to cause a productive infection. It is thus possible that M. tuberculosis H37Ra and M. smegmatis may not be able to cause productive infections because of the continuous triggering of the release of cytokines by the MΦs infected with them. Thus, these infected MΦs acquire bacteriostatic activity and possibly, other phagocytic cells may accumulate around them, leading to granuloma formation. The inherent cytotoxicity of M. tuberculosis H37Rv for MΦs may also be responsible for the diminished elaboration of cytokines (Falcone et al., 1994). As various M. tuberculosis field isolates are known to differ in their virulence and pathogenicity, in the present studies we considered it expedient to include both avirulent and virulent, and non-pathogenic and pathogenic mycobacteria.
The heat-killed mycobacteria, as compared to their live counterparts, induced the infected PMs for enhanced elaboration of IL-6. The live mycobacteria are known to inhibit phagosome-lysosome fusion, whereas heat-killed ones do not (Barker et al., 1997), possibly because of the enhanced secretion of sulphatides by live mycobacteria as compared to the heat-killed bacilli (Gordon et al., 1980). Further, PI3P is known to be retained on the phagosomes which contain heat-killed mycobacteria, but is continuously eliminated from those having live bacilli (Vergne et al., 2005). The ability of the heat-killed mycobacteria to induce more cytokines may also partially reflect the loss of direct cytotoxic effect produced by the viable mycobacteria on infected MΦs (Falcone et al., 1994). Higher IL-6 production by heat-killed mycobacteria may simply reflect that once killed, these mycobacteria may lose partly or completely some of their heat-labile virulence components, and thus may allow greater phagosome-lysosme fusion in infected MΦs.
The human relevance of these studies remains an important point to ponder. Apparently, there is no specific experimental or clinical evidence(s) to suggest the extrapolation of the results of cytokine elaboration, especially IL-6, following murine MΦ and mycobacteria interaction, in vitro, to human situation. However, there are several reports of the interaction of human MΦs and mycobacteria (Beltan et al., 2000; Blanchard et al., 1991), which show results similar to those reported herein. Therefore, it seems plausible to objectively extrapolate the results of this study to humans both for clinical and latent TB, albeit after expedient extended studies, both in murine and human systems. Nevertheless, the extrapolation of results generated in vitro using an isolated population of cells may just be a gross oversimplification of the events occurring in vivo, and thus may require a more complex culture system or a suitable animal model, which should mimic situation in human TB patients, as closely as possible. Additionally, appropriate translational researches are warranted to make these studies applicable to humans.
In conclusion, our results demonstrate that the interaction of PMs with mycobacteria, in vitro, invariably, ensued in the augmented elaboration of IL-6 only, and thus, it may be used as a potential biomarker (stand-alone) or as a biosignature (along with other cytokines), of mycobacterial infection. Such a biomarker/biosignature is warranted to aid in the detection of mycobacterial infection, and also, possibly, to give a fillip to the faster progression of the potential anti-TB drugs presently stagnating in the clinical trial pipe-line, albeit after due caution and skepticism.